Enhancing drought stress tolerance and growth promotion in chiltepin pepper (Capsicum annuum var. glabriusculum) through native Bacillus spp.

The drought can cause a decrease in food production and loss of biodiversity. In northern Mexico, an arid region, the chiltepin grows as a semi-domesticated crop that has been affected in its productivity and yield. An alternative to mitigate the effect of drought and aid in its conservation could be using Plant Growth-Promoting Bacteria (PGPB). The present study evaluated the capacity of native Bacillus spp., isolated from arid soils, as PGPBs and drought stress tolerance inducers in chiltepin under controlled conditions. Chiltepin seeds and seedlings were inoculated with native strains of Bacillus spp. isolated from arid soils, evaluating germination, vegetative, and drought stress tolerance parameters. The PGPBs improved vegetative parameters such as height, stem diameter, root length, and slenderness index in vitro. B. cereus (Bc25-7) improved in vitro survival of stressed seedlings by 68% at −1.02 MPa. Under greenhouse conditions, seedlings treated with PGPBs exhibited increases in root length (9.6%), stem diameter (13.68%), leaf fresh weight (69.87%), and chlorophyll content (38.15%). Bc25-7 alleviated severe water stress symptoms (7 days of water retention stress), and isolates B. thuringiensis (Bt24-4) and B. cereus (Bc25-7, and Bc30-2) increased Relative Water Content (RWC) by 51%. Additionally, the treated seeds showed improved germination parameters with a 46.42% increase in Germination Rate (GR). These findings suggest that using PGPBs could be an alternative to mitigate the effect of drought on chiltepin.

The drought is a problem that affects various regions of the world.It is one of the main limitations of modern agriculture, causing severe physiological stress in plants and compromising food production, as well as the loss of biodiversity [1][2][3][4] .In the last decade, global economic losses in the agricultural sector reached 37 billion dollars 5 .The northern region of Mexico is highly vulnerable to water deficits 6 .experiencing prolonged drought conditions in the extreme and exceptional categories from July 2020 to 2022 7 .Thus, drought threatens the productivity of chiltepin in Chihuahua, Mexico.Capsicum annuum var.glabriusculum, commonly known as chiltepin, is a semidomesticated chili pepper of gastronomic and cultural significance in Mexico 8 .Chiltepin is valued for its high genetic variability, translating into broad adaptability to several environmental conditions.Therefore, chiltepin represents a valuable reservoir of primary genes to improve pepper crop cultivation through enhanced resistance to phytopathogens, pests, and unfavorable abiotic conditions 9 .Thus, it is essential to develop preservation strategies to protect the biodiversity of wild species 10 .In addition, chiltepin has a high economic value (up to 1,200.00 pesos per kilogram 11 .and recently its demand has increased.
A sustainable alternative to mitigate drought stress effects in agriculture is the crop treatment with Plant Growth-Promoting Bacteria.PGPBs act through many interconnected and synergistic mechanisms of action.Among these are phytohormones production, siderophore production, ammonium production, phosphate solubilization, osmoprotectant production, and gene expression regulation associated with antioxidant activity [12][13][14][15] .
The PCA analysis of vegetative parameters from the in vitro germination promotion trial, conducted to determine the best performance as PGPB, explains 86.6% of the variability in the data.PC1 explained 63.2% of the variation, and PC2 explained 23.4% of the variation in the data (Fig. 1).According to the k-means cluster analysis, the group with the most positive effect on growth consisted of the bacteria Bs22-3, Bc30-1, Bt24-4, Bt22-1, Bc16-4, and Bc16-5, corresponding to the parameters of FW, height, root length, and stem diameter.Otherwise, bacteria Bc25-3, Bc25-7, Bc30-2, and the commercial control, showed moderate performance in

Drought tolerance under in vitro conditions
Drought tolerance potential of Bacillus isolates Before being used as inducers of tolerance, the strains resistance to water stress was evaluated under controlled conditions using the PEG test.A minimum survival rate of 60% relative to the control (P0) was considered to classify tolerant bacteria.Thirty percent of the isolates were sensitive to P30, while 38% exhibited high tolerance (Table 5).Treatments Bt25-6 and Bc30-2 were entirely tolerant for the highest evaluated concentration (P50).All isolates remained viable until exposure to P40, except for Bs22-3 and the control strain BsQ.

PEG concentration Tolerant isolates
Tolerant isolates number

Growth promotion
After the in vitro evaluation of germination tests and stress tolerance, two strains of B. cereus (Bc25-7 and Bc30-2) and one strain of B. thuringiensis (Bt24-4) were selected to determine their effectiveness under greenhouse conditions.All isolates induced a significant increase in at least one of the vegetative parameters (Fig. 2, Supplementary Fig. S2).Treatments Bt24-4, Bc25-7, and Bc30-2 increased root length by 9.6% compared to the control (uninoculated seedlings) and 11.5% compared to the commercial isolate.Stem diameter increased by 13.68% with treatments Bt24-4 and Bc25-7 compared to the control and 6.02% compared to the commercial isolate.Likewise, leaf fresh weight increased by 69.87% with isolates Bt24-4, Bc25-7, and Bc30-2 compared to the control.Besides, chlorophyll a content increased by 38.15% in treatments Bt24-4 and Bc25-7 compared to the control and 32.9% compared to the commercial strain.In contrast, parameters such as height, number of leaves, leaf area, stem and root fresh weight, dry weight, chlorophyll b, and carotenoids did not show significant differences compared to the control.Leaf area (cm2) The PCA analysis of vegetative parameters from the greenhouse germination promotion trial, conducted to determine the best performance as PGPB in the greenhouse, explains 91.2% of the variability in the data.PC1 explained 69.0% of the variation, and PC2 explained 22.2% of the variation in the data (Fig. 3).Treatment Bc25-7 exhibited the most positive effect on chiltepin growth across all evaluated vegetative parameters.Otherwise, the isolate Bt24-4 and the commercial control positively affected leaf area, fresh weight, stem diameter, and seedling height, while Bc30-2 mainly increased root length and the number of leaves.

Drought tolerance in greenhouse conditions
The seedlings treated with Bc25-7 presented an alleviation in visual symptoms of wilting caused by severe water stress (loss of leaf turgidity and reduced growth rate) compared to non-inoculated seedlings (negative control), even though the substrate had similar moisture levels (Supplementary Fig. S3).Water stress led to a significant reduction in the relative water content (RWC) in chiltepin leaves.However, all isolates increased RWC by 51.28% compared to the negative control, exhibiting similar behavior to the commercial control (Table 7).Otherwise, applying water stress induced a significant increase in proline content (30 times higher) compared to the unstressed seedlings.However, isolates Bt24-4, Bc25-7, and Bc30-2 showed 50.36% less proline content in leaves compared to the negative control.

Discussion
The morphological and biochemical characterization of native bacterial isolates provides fundamental information about their phenotypic traits, aiding in the initial identification process.Observed characteristics such as colony morphology, color, texture, and spore formation indicated the Bacillus genus 30 .Molecular identification of bacteria by 16S rRNA offers a more precise and discriminatory approach, allowing differentiation of closely related species within the Bacillus genus 31 .Among the isolates identified as members of the B. cereus group, a high  Improving crop germination processes remains critical for increasing productivity and addressing agronomic limitations 32 .Chiltepin, a wild chili species, poses challenges due to its low germination rates and high variability 9,33 .Therefore, the application of Bacillus spp.rhizobacteria in this study demonstrated significant improvements in chiltepin seedling germination parameters, offering a promising solution to these challenges.The findings of this study are consistent with previous research demonstrating the germination-promoting effects of Bacillus spp. in various species within the Capsicum genus.For instance, Bolaños et al. 34 observed a notable increase in germination rates in pepper seeds (C.annuum L.) after inoculation with B. licheniformis M2-7, while B. subtilis was found to enhance germination in "Kandil Dolma" peppers 35 .These observed improvements can be attributed to the production of phytohormones such as gibberellins, cytokinins, and auxins by Bacillus spp. 33.These growth regulators play fundamental roles in seed germination by stimulating nutrient mobilization, hydrolyase synthesis, and early root development through cell division and enzymatic actions 36,37 .The significant reduction in mean germination time indicates accelerated germination, which is crucial, especially under stress conditions, as it increases the likelihood of successful seedling establishment and growth 32 .The observed reduction in MGT aligns with previous research, as reported by Sosa et al. 38 and Dutta et al. 39 , who documented similar reductions in C. chinense Jacq.and C. frutescens L. seeds inoculated with Bacillus spp.
Furthermore, vigor and germination indices are essential indicators of seed potency and potential yield during germination 32,40 .The results found in this study were consistent with studies reported by Jayapala et al. 41 , who observed an increase in vigor and germination indices in chili plants through biopriming with Bacillus sp.BSp.Finally, the slenderness index, which reflects the quality and resilience of seedlings, revealed robust seedlings in a significant proportion of treatments after applying Bacillus spp. 42.These findings suggest that Bacillus spp.isolates accelerate germination and facilitate robust seedling establishment by promoting root development and rapid shoot growth.
The positive effects observed in the growth of chiltepin plants in the greenhouse trial align with previous studies demonstrating the beneficial impact of Bacillus spp. on various plant species.The treated seedlings exhibited increased root length, stem diameter, leaf fresh weight, and chlorophyll content, indicating improved vegetative development and potential yield enhancement.This corroborates the findings of García et al. 29 , Kazerooni et al. 27 , and others who reported significant increases in root length and fresh weight in chili and other plant species after inoculation with Bacillus strains 27,29,43,44 .Such morphological improvements are crucial for enhancing nutrient uptake and overall plant vitality, impacting crop productivity 45 .Furthermore, increased chlorophyll content observed in treated chiltepin plants suggests enhanced photosynthetic activity, which is crucial for meeting elevated metabolic demands during critical stages such as flowering and fruiting 46,47 .
The underlying mechanisms behind these growth-promoting effects likely involve multiple pathways facilitated by rhizobacteria.Notably, the capacity to solubilize essential nutrients such as phosphorus and zinc from their non-assimilable forms into accessible forms represents a fundamental aspect of plant-microorganism interactions 48,49 .The solubilization of phosphorus and zinc by Bacillus spp.can be attributed to various mechanisms, including medium acidification, secretion of organic acids, and enzymatic activities, all of which contribute to improving nutrient availability in the rhizosphere 50,51 .Additionally, the production of ammonia by rhizobacteria serves as a vital nitrogen source for plants, stimulating root development and overall biomass accumulation 52,53 .The positive impact of ammonia production on chili plant growth observed in this study corroborates previous findings by Bhattacharyya et al. 52 , and Abdelwahed et al. 54 .regarding the role of Bacillus spp. in nitrogen supply and plant development.Furthermore, the production of siderophores by Bacillus strains represents another mechanism contributing to plant growth promotion.Siderophores enhance plant iron availability, aiding nutrient uptake and serving as a competitive advantage against phytopathogens 27,55 .The isolation of native Bacillus strains producing siderophores in this study suggests their potential as biocontrol agents and growth promoters in agricultural systems.Additionally, the production of exopolysaccharides by Bacillus spp.has significant implications for soil health and plant-microorganism interactions [56][57][58][59] .EPS plays various roles, including soil aggregation, water retention, and nutrient enrichment, ultimately facilitating root colonization and environmental stress mitigation in plants 13,60 .The growth-promoting traits the native Bacillus isolates tested underscore their potential as biofertilizers in chiltepin chili production systems.By enhancing nutrient availability and improving soil properties while mitigating environmental stress, these types of rhizobacteria offer sustainable alternatives to conventional fertilizers and pesticides, thus contributing to ecological balance and horticultural crop productivity 53,61 .
The rhizobacteria isolates displayed high tolerance up to an osmotic pressure of −1.75 MPa.Previous studies have reported varying degrees of drought tolerance in Bacillus spp.under similar conditions 62 For instance, Ashry et al. 63 reported that 25% of isolates isolated from arid soils showed resistance to drought under different water potentials ranging from −0.15 to −1.2 MPa.In another study, B. megaterium and B. licheniformis were able to grow at the lowest water potential tested (−0.73 MPa 13 .This adaptation to water deficit is attributed to the genus ability of Bacillus to form endospores 63 .Other authors associate the resistance of Bacillus spp.and the amelioration of drought stress in plants with mechanisms such as EPS production, phytohormones, enzymes, and osmolytes 13,62 . .The findings of this study provide insights into the underlying mechanisms of drought tolerance potential of Bacillus isolates and their effects on chiltepin seedlings under stress conditions.The observed results demonstrate the ability of Bacillus isolates to enhance drought tolerance in chiltepin seedlings, as evidenced in both in vitro and greenhouse experiments.The in vitro evaluation of stress induction in chiltepin seedlings revealed that specific Bacillus treatments significantly improved seedling survival rates under stress conditions.Treatments such as www.nature.com/scientificreports/Bc25-3 and Bc25-7 showed notable increases in survival rates compared to the negative control (non-inoculated plants).This improvement in survival can be attributed to the ability of Bacillus isolates to induce stress tolerance mechanisms in plants.Similar findings have been reported in previous studies, where Bacillus treatments increased survival rates of various plant species under drought conditions [64][65][66] .Furthermore, the morphological characteristics of stressed seedlings treated with Bacillus isolates improved significantly compared to the negative control.Treatments such as Bc24-4, Bc25-3, Bc25-4, Bt25-6, Bc25-7, and Bc30-2 showed enhancements in height, stem diameter, and slenderness index, indicating a positive impact on seedling growth and development.These findings are consistent with previous research demonstrating the beneficial effects of Bacillus treatments on plant morphology under drought stress 67,68 .Additionally, the greenhouse trial confirmed the effectiveness of Bacillus treatments in mitigating drought stress in chiltepin seedlings.The Bc25-7 treatment alleviated visual wilting symptoms and significantly increased the relative water content (RWC) in chiltepin leaves compared to uninoculated plants.According to categories described by Bandurska 69 , unstressed plants have an RWC in leaves above 90%, while plants under mild drought stress show values between 60 and 70%.An RWC between 40 and 60% indicates moderate water stress, and values below 40% correspond to severe stress.Thus, the treatment with Bacillus spp.they reduced the stress level in chiltepin seedlings from severe to moderate.These findings suggest that the tested native Bacillus isolates enhance drought tolerance in chiltepin seedlings by modulating physiological responses.Similar results have been reported in previous studies, where Bacillus treatments led to an increase in RWC and an increment in proline accumulation in plants subjected to drought stress 70,71 The underlying mechanisms behind the observed effects of Bacillus treatments on drought tolerance in chiltepin seedlings are likely multifaceted.Previous research has suggested several potential mechanisms, including the production of bacterial biofilms, modulation of stomatal behavior, and activation of adaptive responses mediated by phytohormones such as abscisic acid (ABA) 13,64 .Additionally, Bacillus treatments may enhance plant antioxidant activity, gene expression, and synthesis of protective proteins, contributing to stress tolerance overall 72 .It is important to note that no uniformity rates were observed in the performance of the native isolates examined in the experiments.The disparity in their effectiveness as PGPB could be attributed to genetic and environmental factors.According to Samain et al. 73 , the effectiveness of the strains depends on their ability to establish specific interactions with plants and their level of adaptation to adverse conditions.This is the first study to conduct a comprehensive assessment of the potential of Bacillus genus rhizobacteria as promoters of plant growth and mitigators of water stress in chiltepin seedlings (Capsicum annuum var.glabriusculum).The ability of Bacillus isolates to promote chiltepin seed germination and growth was demonstrated both in vitro and in vivo.Improvements in germination parameters such as germination rate and speed, as well as vigor and germination index, indicate the potential of these isolates as biostimulants for chiltepin production.Evaluation of drought tolerance revealed that Bacillus isolates could mitigate the adverse effects of water stress on chiltepin seedlings.Both under in vitro conditions and in the greenhouse, Bacillus treatments improved seedling survival, reduced visual symptoms of stress, and increased relative leaf water content.Together, these findings highlight the potential of Bacillus genus rhizobacteria as practical tools for enhancing chiltepin crop production and resilience, providing sustainable alternatives to conventional agricultural inputs, and contributing to food security and environmental sustainability in the study region.However, further studies are needed to fully understand the underlying mechanisms and optimize the application strategies of these biostimulants in field agricultural production systems.

Soil sample collection
Representative soil samples were collected from 15 commercial chili plots from the agricultural zone in Meoquí, Chihuahua, Mexico (28°23´23″ N, 105°37´25″ W) in January 2022.The samples were labeled and transferred to 4 °C for processing.

Isolation and growth of Bacillus spp.
The isolation process of Bacillus spp. was carried out in the Applied Microbiology, Phytopathology, and Postharvest Physiology Laboratory (MAFFP) of the School of Agrotechnological Sciences of the Autonomous University of Chihuahua (UACH).The bacteria were isolated according to the methodology described by Astorga et al. 74 .For this, 0.5 g of soil was dissolved in 4.5 mL of 0.85% (w/v) sterile saline solution and kept under constant agitation for 3 min using a vortex.Subsequently, a heat treatment at 80 °C was applied for 10 min in a water bath, followed by serial micro-dilutions in a microplate up to a 10 -6 dilution factor.Colonies showing the typical macroscopic characteristics of the Bacillus genus were subcultured until pure colonies were obtained.

Morphological and biochemical identification of Bacillus spp.
For the identification of the isolates, the hanging drop motility test was conducted according to the methodology of Covadonga & de Silóniz 30 , Gram staining, endospore staining using the Schaeffer-Fulton technique, and potassium hydroxide (KOH) test were performed.In addition, microscopic characteristics and colonial morphology were reviewed as described by Garrity et al. 75,76

Molecular identification of Bacillus spp.
A polymerase chain reaction (PCR) was carried out to amplify the 16S rRNA fragment for molecular identification.Total genomic DNA extraction from the rhizobacteria was performed using the modified phenol-chloroform method, according to Bardakci & Skibinski 76 .The concentration and purity of DNA from the samples were determined with a biospectrophotometer using the SW Version: 4. www.nature.com/scientificreports/prepared with the following components from Thermo Scientific (Massachusetts, USA): 1 × Taq buffer with KCl, 2.5 mM MgCl2, 0.2 mM dNTP'S, 0.25 U Taq DNA polymerase, and H2O.Subsequently, 2.5 μL of primers 27F-5' AGA GTT TGA TCC TGG CTC AG 3' and 1492R-5' TAC GGT TAC CTT GTT ACG ACT T 3' (Integrated DNA Technologies, Iowa, USA) and DNA at a concentration of 10 ng/μL were added.The PCR was performed with the following amplification program: an initial denaturation for 10 min at 95 °C followed by 30 amplification cycles (1 min of denaturation at 94 °C, 1 min of annealing at 50 °C, and 1.5 min of extension at 72 °C) followed by a final 10-min extension at 72 °C77 , The amplified fragments were sent to Macrogen Crop (Rockville, Maryland, USA) for sequencing using the Sanger method.The sequences were submitted to the NCBI (National Center for Biotechnology Information) to obtain the accession number.Finally, a search for homologous sequences was conducted using the nBLAST tool from the gene bank.

Seed inoculation
Seeds of C. annuum var.glabriusculum were used, extracted from wild plants from the locality of Chínipas, Chihuahua (27 24′ 0 N, 108 32′ 0 W).They were selected and disinfected in a 5% (v/v) NaClO solution under constant agitation for 5 min, followed by 3 rinses with distilled water (H 2 Od).They were then kept in sterile H 2 Od for 24 h and treated with bacterial solutions (1 × 10 8 CFU/mL) under constant agitation at 150 rpm / 28 °C for 1 h, after which the bacterial solution was discarded.The plates were placed in a germination chamber with a photoperiod of 16 h of light at 28 °C and 8 h of darkness at 25 °C.

Germination parameters
To determine the effect of the isolates on germination promotion, 25 chiltepin seeds were inoculated with 12 Bacillus spp.isolates.The commercial isolate QST 713 of B. subtilis (Serenade® ASO = BsQ) was used as a positive control, and seeds without treatment were used as a negative control (n = 4).Germination was monitored 19 days after sowing (DAS) to determine germination parameters and indices (Table 8).In addition, vegetative parameters (fresh weight, stem length, root, stem diameter) were evaluated in 5 seedlings.

Beneficial plant growth promoting traits
The solubilization of phosphorus, ammonia production, siderophore production, zinc solubilization, and exopolysaccharide production were evaluated in the 12 isolated Bacillus spp.

Phosphorus solubilization
The phosphorus solubilization capacity of the Bacillus spp.isolates were assessed by inoculating 5 μL of growth (1 × 10 8 CFU/mL) on Pikovskaya medium (n = 5).The solubilization index (SI) was determined according to the following formula 80 .

Ammonium production
Ammonia production was evaluated using a colorimetric method through the Nessler assay 81 adapted to a microplate adjusting to a total volume of 200 μL (n = 4).

Drought tolerance potential of Bacillus isolates
To assess the resistance of Bacillus spp.isolates to different concentrations of polyethylene glycol, the technique described by Yadav et al. 85 was used.For this, a microplate with 180 μL of nutrient broth culture medium supplemented with polyethylene glycol at concentrations of 10, 20, 30, 40, and 50% was used, along with control without PEG (P10, P20, P30, P40, P50, and P0, respectively).5 mL of each bacterial growth (1 × 10 8 CFU/mL) was centrifuged at 5000 rpm, 4 °C for 5 min.The supernatant was then decanted, and the pellet was resuspended in 0.5 mL of 0.85% sterile saline solution.Next, the wells were inoculated with 20 μL of the bacterial suspension and kept in an incubator at 28 °C for 96 h (n = 3).To assess bacterial growth, an OD measurement at 600 nm was taken, and the survival rate of each bacterium was calculated relative to the control (P0).To qualitatively evaluate viability, 10 μL of resazurin (0.03 M) was applied to each well; a color change from blue to pink shades indicated cell viability 86 .

In vitro evaluation of stress induction in chiltepin seedlings
Seedlings 23 days after sowing (DAS) inoculated with the bacterial solutions (1 × 10 8 CFU/mL) were transferred to tubes containing 1 mL of PEG (30%) (n = 9).After 5 days, the survival rate of seedlings and vegetative parameters such as height, root length, stem diameter, and slenderness index were evaluated as described in the previous sections.

Growth promotion and drought tolerance in greenhouse conditions
In the growth promotion and drought tolerance assays under greenhouse conditions, the strains Bt24-4, Bc25-7, and Bc30-2 were used.These strains were selected due to their outstanding performance in the germination and in vitro water stress assays.During the germination of chiltepín, they showed the best results in GR, the germination index, and GSI.Additionally, these strains were grouped within the two highest-performing groups in analyzing vegetative parameters using PCA-cluster analysis.Moreover, they also induced a high survival rate and a low slenderness index in chiltepín seedlings in the in vitro water stress experiment.

Growth promotion
For this assay, seedlings were produced from disinfected seeds, and transplanting took place 60 days after sowing (DAS) with 3-4 true leaves.The seedlings were placed in a greenhouse, and temperature and humidity were monitored throughout the experiment.Irrigation was carried out every 48-72 h with distilled water (H 2 Od,) and a Steiner nutrient solution (pH 6.0; EC 1250 µS) was applied 87 .The first inoculation was done on the day of transplant by applying 5 mL of a bacterial suspension (1 × 10 8 CFU/mL) to the base of each seedling, and this was repeated three times every 15 days.Serenade® was used as a positive control, and seedlings treated with water were used as a negative control (n = 6).The evaluation of vegetative parameters was carried out 37 days after transplant (DAT), considering height, leaf count, leaf area 88 , stem diameter, root length, fresh and dry biomass, pigment content (chlorophyll a, b, and carotenoids) 89

Drought tolerance in greenhouse conditions
This experiment used the commercial product Xp Amino® and the Serenade® isolate as positive controls.Otherwise, seedlings without treatment were used as a negative control, and seedlings without treatment and stress were used as an absolute control (n = 6).75 DAS seedlings inoculated with Bacillus spp.isolates were subjected to water stress by withholding water for seven days 72 .To do this, the seedlings were watered to saturation, submerging the vessels for 30 s in 500 mL of tap water.Excess water was then decanted, and after 1 h, the substrate's moisture was determined (3 Way Soil Meter).The leaves proline content and relative water content (RWC) were determined for the evaluation.The total proline estimation was carried out using the method of Bates et al. 90 ; 0.5 g of leaf tissue was homogenized in 10 mL of 3% aqueous sulfosalicylic acid and passed through Whatman Filter Paper 1. Next, 2 mL of the filtrate was taken, and 2 mL of concentrated glacial acetic acid and 2 mL of acid ninhydrin (1.25 g ninhydrin, 30 mL glacial acetic acid, and 20 mL 6 M phosphoric acid) were added.The mixture was placed in a water bath at 100 °C for 1 h, and the reaction was stopped on ice.Subsequently, 4 mL of toluene was added, and the absorbance was read at 520 nm.The total proline content estimation (µM/g of fresh plant material) was made using a standard L-proline curve.The relative water content (RWC) in the leaves was determined using the following formula 91 : where: FW is the fresh weight after seedling harvesting, WT is the weight of leaves saturated when submerged in H 2 O for 4 h (turgid weight), and DW is the constant dry weight.

Table 1 .
Sequence analysis by 16S rRNA fragment and similarity with homologous sequences available in the NCBI GenBank.

Table 3 .
Germination parameters (GR, MSG, MGT, and GSI) and vigor, germination, and slenderness indices in C. annuum var.glabriusculum seeds treated with Bacillus spp.The average of 4 repetitions represents the data.Different letters indicate significant statistical differences according to the Scott Knott test (p ≤ 0.05).

Table 4 .
Vegetative parameters (height, root length, stem diameter, FW, and slenderness index) of C. anuum var.glabriusculum seedlings treated with Bacillus spp. on 19 DAS.The average of 4 repetitions represents the data.Different letters indicate significant statistical differences according to the Scott Knott test (p ≤ 0.05).

Table 6 .
Survival rate and vegetative parameters (height, root length, stem diameter, and slenderness index) of C. annuum var.glabriusculum seedlings under water stress (PEG 30%).The average of 9 repetitions represents data.Different letters indicate significant statistical differences according to the Scott Knott test (p ≤ 0.05).TreatmentSurvival rate (%

Table 7 .
RWC and proline in C. annuum var.glabrisculum leaves subjected to drought stress.Data are represented by the average of 6 and 3 repetitions, respectively.Different letters indicate significant statistical differences according to the Scott Knott test (p ≤ 0.05).sequencesimilarity with reference strains further supports their classification.Specifically, isolates Bc25-3 and Bc30-1 exhibited complete similarity to B. cereus reference sequences, indicating a close phylogenetic relationship.In contrast, isolate Bs22-3 showed 100% similarity with B. subtilis, diverging from the predominant group of B. cereus.Furthermore, identifying isolate Bw19-1 as B. wiedmannii was less studied than other Bacillus species.B. wiedmannii isolation represents a notable contribution to understanding the microbial diversity of arid soil areas.

Table 8 .
Germination parameters and indices.Zinc solubilizationThe zinc solubilization capacity was evaluated on Bunt & Rovira 83 plate culture medium supplemented with 0.1% ZnO as an insoluble zinc source (n = 5).